Recent developments in fluorescence detection coupled with bioconjugation techniques are leading to a rapid proliferation of advanced fluorescence-based techniques in chemistry and the life sciences, such as fluorescence microscopy, flow cytometry, versatile biological assays, and biosensors. These fluorescence techniques make extensive use of organic dye molecules as probes. However, intrinsic limitations of the conventional dyes, such as low absorptivity and poor photostability, have posed difficulties in further developments of high-sensitivity imaging techniques and high-throughput assays. A number of strategies for developing brighter fluorescent probes have been pursued. For example, luminescent nanoparticles such as inorganic semiconductor quantum dots (Qdots) are under active development and now commercially available from Life Technologies (Invitrogen). (Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P, Science 1998, 281, 2013-2016. Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544.) An alternative fluorescent nanoparticle is dye doped latex spheres, which exhibit improved brightness and photostability as compared to single fluorescent molecules because of multiple dye molecules per particle and the protective latex matrix. (Wang, L.; Wang, K. M.; Santra, S.; Zhao, X. J.; Hilliard, L. R.; Smith, J. E.; Wu, J. R.; Tan, W. H. Anal. Chem. 2006, 78, 646-654).
The limitations of current luminescent particles provide a need for exploring alternative strategies for the design of more highly fluorescent nanoparticles. Recently, fluorescence semiconducting polymer dots (Pdots) have attracted interest because of their fluorescence brightness and photostability as compared to Qdots and dye-loaded latex beads. The use of fluorescent polymer dots as fluorescent probes also can confer other useful aspects. Recently, surface functionalization has been achieved by a co-condensation scheme where amphiphilic polymer molecules bearing functional groups were blended with semiconducting polymers to form Pdots with surface reactive groups. Bioconjugation has been demonstrated by reacting the functional groups with biomolecules, and the Pdot-bioconjugates can specifically and effectively label biomolecules for cellular imaging, bioorthogonal labeling, and in vivo tumor targeting.
However, there can be drawbacks when the current Pdots are used as fluorescent probes in practical applications. Many biological applications can include detecting multiple targets simultaneously; thus, there is a need for probes that possess narrow-band emission peaks for spectral multiplexing. However, currently available Pdots can exhibit very broad emission spectra, which limit their usefulness in practical applications. The spectral width of a fluorescent probe can be characterized by the full width at half maximum (FWHM) of its emission peak. In general, currently available Pdots exhibit broad emission spectra with large FWHMs. Such broad emission spectra are a drawback for multi-target detection in biology. Therefore, there is a need to design and develop new type of Pdots with narrow-band emissions.